Development of an Automated, High-Throughput Bactericidal Assay That Measures Cellular Respiration as a Survival Readout for Neisseria meningitidis

Abstract

Complement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity against Neisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.

Serum bactericidal antibody assays – The role of complement in infection and immunity

“…An automated, high-throughput kinetic bactericidal assay based on the fluorescent respiration product of resazurin has been developed [91]. This reduces assay volume, shortens assay time and facilitates the automation of data analysis.…”

A review of complement sources used in serum bactericidal assays for evaluating immune responses to meningococcal ACWY conjugate vaccines

“…Of note, a modified version of the hSBA assay was developed in recent years; this high-throughput, automatable method involves using a colored indicator of cell metabolic activity in a liquid medium that can be correlated with hSBA titers. 38 Assay results have been demonstrated to correlate with those from conventional hSBA assays, 38 and some recent analyses have used this modified assay. 39,40 Serogroup B vaccines A 1983 publication demonstrated that subjects with high rSBA titers against serogroup B meningococcal strains had much lower or even nonexistent bactericidal activity to the same strains in hSBA assays.…”

Immunogenicity and safety of investigational vaccine formulations against meningococcal serogroups A, B, C, W, and Y in healthy adolescents

“…Analysis of the fluorescence readouts and computation of high-throughput hSBA titers was conducted as described previously. 34 The reciprocal serum dilution that resulted in a 50% reduction of fluorescence (IC 50 ) relative to the normalized maximum signal was reported as the high-throughput hSBA titer of the sample. Immune responses to N. meningitidis serogroup Y were assessed using the conventional hSBA assay.…”

“…Sera were analyzed at the Novartis Clinical Laboratory Sciences (Marburg, Germany). Immune responses to N. meningitidis serogroups ACW and serogroup B test strains were assessed using a high-throughput serum bactericidal assay with human complement (hSBA) as described previously 34 with some modifications. In brief, the high-throughput hSBA assay adheres to the same principles as the conventional hSBA assay but differs in that the bacterial outgrowth step is in a liquid suspension containing the cell-permeable redox substrate resazurin dye (the active ingredient of alamarBlue Ò ).…”